| Maltoheptaose is a kind of linear maltooligosaccharide of specific length that is connected by seven glucose units throughα-1,4 bonds.Compared with maltotriose and tetrasacchride,it has a lower osmotic pressure,higher viscosity,better moisturizing effect and stronger film-forming performance.In addition,maltoheptaose has the function of embedding small molecules.It can be applied to food,medcine,cosmetic and other fields.Enzymatic preparation of maltoheptaose has advantages of the mild reation conditions and environmetal protection.And it is becoming the research focus.Cyclodextrinases can catalyzeα-1,4 bonds ofβ-CD,occuring ring-opening reaction,producing maltoheptaose.In this study,by multiple alignment and structure simulation,two cyclodextinase genes derived from Thermococcus sp.B1001 and Thermococcus litoralis were selected for recombinant expression in E.coli.And the enzymatic properties,the performance and condition optimization of maltoheptaose preparation were characterized.Then,the cyclodextrinase gene derived from Thermococcus sp.B1001 was recombinantly expressed in B.subtilis,protein purification and preparation of maltoheptaose.Finally,the recombinant B.subtilis shake flask optimization and 3-L tank fermentation.The main results were listed as follows:（1）Gene mining,recombinant expression and enzymatic properties:multiple alignment and enzyme characterization indicated that N-terminal sequence of high-spcificity cyclodextrinases were longer than ordinary cyclodextrinases.Cyclodextrinases with N’domian could form complete catalytic binding groove with enzyme molecules and could hydrolyze cyclodextrin without oligomerization.In addition,the N’domian restricted the binding of long-chain molecules such as starch and could enhance the specific hydroluysis of cyclodextrin.Two cyclodextrinase genes derived from Thermococcus sp.B1001 and Thermococcus litoralis were successfully cloned and expressed in E.coli.The specific activities of TsCDase and TlCDase after heat treatment and nickel column purification were1208.04 U·mg-11 and 409.06 U·mg-1.The optimal temperature of TsCDase and TlCDase were90℃.The optimal pH of TsCDase and TlCDase were 5.5 and 6.0.The half-life were 120 min and 60 min at 90℃.And the stability were the best under the conditions of pH 8.0 and 6.5.Among five substrates ofα-cyclodextrin,β-cyclodextrin,γ-cyclodextrin,soluable starch and pullulan,the optimal substrates of TsCDase and TlCDase wereβ-cyclodextrin andγ-cyclodextrin.（2）Characterization and condition optimization of maltoheptaose preparation of cyclodextrinase:optimization of enzyme amount addition,reaction time,initial pH,substrate concentration determined the optimal enzyme conversion conditions:the yield of maltoheptaose was 81.19%and 85.95%,accounting for 95.24%and 92.92%of all maltooligosaccharides,when adopting the following conditions:a dosage of 80 g·L-1β-cyclodextrin,pH 5.5 and 6.0,90℃,25 U per gramβ-cyclodextrin,incubated for 4 hours.（3）Recombinant expression,purification and application of cyclodextrinase gene tscd in B.subtilis:recombinant B.subtilis WS9/pUB110-tscd was constructed.The intracellular enzyme activity reached 5.9 U·mL-1 after 24 hours of fermentation in the shake flask.SDS-PAGE indicated that the target band appeared at 66 kDa,indicating that tscd was successfully expressed in B.subtilis.The specific activity was 637.95 U·mg-1 after Ni-NTA purification.And the yield of maltoheptaose was 82.33%,accounting for 94.55%of all maltooligosaccharides,when adopting the following conditions:a dosage of 80 g·L-1β-cyclodextrin,pH 5.5,90℃,25 U per gramβ-cyclodextrin,incubated for 4 hours.（4）Fermentation optimization of recombinant B.subtilis:the fermentation conditions of the recombinant bacteria were optimized.The optimum medium after shake flask optimization were 10 g·L-1 corn syrup from Angel,10 g·L-1 yeast extract,5 g·L-1 glycerol,Mg2+1 mmol·L-1,K2HPO4·3H2O 16.43 g·L-1,KH2PO4 2.31 g·L-1,and the activity of the recombinant bacteria reached 13.93 U·mL-1.3 L tank fermentation was carried out based on the result of the optimization of shake flask.And the carbon source in the feed medium was400 g·L-1 glucose,the nitrogen source were 50 g·L-1 corn syrup from Angel and 50 g·L-1 yeast extract.The enzyme activity reached the maximum value after 70 hours of fermentation.The total enzyme activity was 481.75 U·mL-1.The intracellular enzyme activity was 390.83U·m L-1,and the extracellular enzyme activity was 90.92 U·m L-1.The total activity was about34.58 times of the optimized result of shake flask. |
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